Ruriko Haranaka

Role of first stimulating agents in the production of tumor necrosis factor.

SummaryThe conditions and kinetics of tumor necrosis factor (TNF) production were examined. For TNF production, dual stimulation is necessary. Priming agents such as BCG, Corynebacterium parvum, and zymosan, which can stimulate the reticuloendothelial system (RES), are good substances for TNF production with the aid of lipopolysaccharide. Wide differences are observed in TNF producibility among different priming agents. The producibility of TNF depends on the degree of stimulation of the RES by the priming agents. Those priming agents, e.g., Propionibacterium acnes and Corynebacterium anaerobium, that are able to induce substantial RES hyperplasia are also able to induce high levels of TNF activity. Following administration of large doses of BCG or zymosan, mice were found to produce TNF activity. However, PPD, OK 432, PSK, and Choreito were unable to induce TNF activity.

Traditional Chinese Medicines and Drugs in Relation to the Host-Defense Mechanism

Pseudomonas aeruginosa infection is observed mostly under immunosuppressive conditions. We have clarified that the combined use of antibiotics and specific antibodies is effective against mouse P. aeruginosa infection in vivo [1–5]. From our previous experiments, traditional Chinese medicines and crude drugs are known to cause stimulation of the reticuloendothelial system [6]. Kiger et al. [7] found that P. aeruginosa treatment (108 killed organisms) had a capacity to induce release of tumor-necrotizing factor in the serum of bacille Calmette-Guerin (BCG)-pretreated mice to the same extent as lipopolysaccharide (LPS) treatment. Our experiments on tumor necrosis factor (TNF) have demonstrated the production of TNF as well as its associated severe toxicity [8–10]. Recently, recombinant human TNF has been mass produced and many actions of this factor have become clear. We have found that by employing traditional Chinese medicines as priming agents, the toxic symptoms after administration of LPS could be reduced.

Pharmacological Action of Hachimijiogan (Ba-wei-wan) on the Metabolism of Aged Subjects

In order to elucidate the active mechanism of traditional Chinese medicine, we investigated the effects of Hachimijiogan which has been alleged to restore the function of old degenerated organs. After administering Hachimijiogan to aged rats and mice, we examined the lipid and glutathione metabolism. The cholesterol and triglyceride levels in the mice liver were significantly decreased in the Hachimijiogan treated group. The cholesterol turnover was examined using labeled cholesterol in aged mice, and Hachimijiogan may increase the turnover of cholesterol metabolism. The lenticular glutathione levels and enzyme activities concerned with glutathione metabolism were investigated in aged rats given Hachimijiogan, suggesting that Hachimijiogan may offer protective and therapeutic effects against senile cataract. From our study, Hachimijiogan is considered to improve the lipid and glutathione metabolism as a result of its complicated in vivo compound effects.

Preventive effect of several drugs against Pseudomonas aeruginosa infection and the toxicity of combined tumor necrosis factor with lipopolysaccharide: relationship between lethality and the arachidonic cascade.

The participation of tumor necrosis factor (TNF) and lipopolysaccharide (LPS) inPseudomonas aeruginosa (Pa) infection was examined. The lethal challenge of Pa or TNF and LPS injection could be prevented by pretreatment with anti-TNF antibody, polymyxin B, ONO 1078, or Shosaiko-to. The combined effects of TNF and LPS may be deeply related to the lethality of Pa infection. The activities of leukotriene(LT) C4/D4/E4 or platelet activating factor (PAF) were also related to the lethality of Pa infection, probably due to the subsequently produced TNF which acts in combination with LPS. Activating the host defence mechanism with biological response modifiers like Chinese medicines was effective against Pa infection. One mechanism could involve an activity as an LT inhibitor or PAF antagonist. Following the administration of TNF and/or LPS, the serum levels of arachidonic cascade products underwent various changes. With a combination of TNF and LPS, there was a synergistic increment of prostaglandins, thromboxane, and LT. Following pretreatment with Shosaiko-to, suppression of LTs was dominant even with the combination of TNF and LPS, which might be related to the lethality of the infection or combined TNF with LPS.

Establishment and characterization of human myelomonocytic (TYS) and histiocytic (TYH) cell lines.

Two human hematopoietic cell lines (TYS and TYH) with monocytic characteristics were derived from the peripheral blood of a patient with acute myelomonocytic leukemia and of another with a follicular large‐cell type of malignant lymphoma. The TYS cells, derived from the leukemia patient, revealed a monocytic appearance with microvilli at one side and had many granules and vacuoles. They showed strongly positive reactions with α‐NBE, NASDAE, and AcP, and were reactive with monoclonal antibodies such as Oklal, 12 and B1. The TYS cells, which phagocytized carbon particles and antibody‐coated SRBC but not latex particles, released lysosomal enzymes and tu‐ moricidal factor into the supernatant. The TYH cells, derived from the malignant lymphoma patient, had abundant cytoplasm and pseudopods detectable by electron microscopy with a monocytoid appearance and virus‐like particles in the cytoplasm. They showed strongly positive reactions with α‐NBE, NASDAE and β‐Gase, but no reactivity with monoclonal antibodies or with surface markers except Fc‐γ‐R. TYH cells phagocytized latex particles very well. Two different human monocyte‐histiocyte lineages were thus established. During culture, the TYS and TYH cells maintained their characteristics over 28 and 16 months of Passage, respectively.

Purification characterization and anti‐tumor activity of a new cytokine, histiocyte‐secreted‐factor (HSF)

Purification of cytokines was carried out while monitoring their in vivo anti‐tumor activity and in vitro cytotoxic activities. As a result, purified new cytokines were obtained from culture supernatant of a histiocytic cell line and from rabbit serum. Briefly, a new cytokine (HSF, histiocyte‐secreted‐factor) was purified from the culture supernatant of the histiocytic cell line (TYH) which we established from the peripheral blood of a malignant‐lymphoma patient. The purified samples exhibited suppressive effects on tumor growth but no necrotizing activity towards transplanted murine tumors. The substance displayed no cytotoxic activity against L cells (mouse fibroblast cells). The molecular weight of human HSF was about 42 kDa as estimated by SDS‐PAGE. Amino‐acid sequencing of the purified HSF from the culture supernatant was performed, but the N‐terminal was blocked. Next, a new cytokine was purified from rabbit serum stimulated with Propionibacterium acnes and elicited with lipopolysaccharide. The rabbit HSF was isolated by the same procedures as those used for the human HSF purification steps. Amino‐acid sequencing was carried out after enzyme digestion. Three parts of the amino‐acid sequence of the rabbit HSF were determined as LPPGLLAPMRQLRS‐, NLEXFTNGMEQHYAQL‐, and NPAENQAHELPNQLN‐. A computer‐based homology search demonstrated that these sequences were novel. The molecular weight of HSF as determined using anti‐peptide antibodies revealed the following values: human HSF, 41 and 46 kDa; rabbit HSF, 35, 42 and 55 kDa.